9722356 Bloom The goal of this CAREER plan is to establish a competitive research program while at the same time creating a stimulating learning environment for students in both the research lab and the classroom. Research is aimed at defining the role that local DNA sequence context plays in the efficiency of repair of damaged DNA bases and in the frequency of mutations resulting from unrepaired damage. The long term objective is to elucidate molecular mechanisms governing the formation of mutations in DNA which influence protein-DNA interactions. The premise is based on the nonrandom distribution of mutations throughout a genome, which may be explained by differences in local DNA structure and stability. Local DNA sequence may determine the distribution of mutations in a genome by affecting the efficiency of the DNA-repair enzymes at specific sites. The efficiency of excision of the damaged base, ethenoadenine, by human 3-methyladenine DNA glycosylase (AAG) is examined by a novel fluorescence-based assay, measuring the kinetics of base excision of ethenoadenine by AAG in real time. Escherichia coli uracil DNA glycosylase (UDG) which has a narrow substrate specificity is included for comparison. The influence of local DNA sequence context on mutations due to DNA synthesis errors at template ethenoadenine sites by DNA polymerase III (pol III) and by DNA polymerase delta (pol delta), replicative polymerases of E. coli and eukaryotes, respectively, are studied in order to test whether ethenoadenine may have different miscoding potentials. The educational activities center around integrating research, learning and effective teaching. The overall theme is to use research and discovery as tools to stimulate learning and reinforce concepts taught in the classroom. This is done by including undergraduate and graduate students in accomplishing research objectives and by revising an advanced undergraduate biochemistry lab curriculum to incorporate a more "research-like" approach that facilita tes discovery based learning. Additional participation is also attempted in two multidisciplinary graduate research training programs, which are designed to provide students with broad educational scopes and to encourage them to go beyond traditional boundaries of a given discipline in their approach to research problems and goals. This type of training is essential for preparing students for a wide range of career opportunities and changing job markets. %%% The goal of this career plan is to establish a competitive research program while integrating classroom teaching and laboratory research into a stimulating learning environment. Research activities are aimed at defining the role of DNA structure in the formation of mutations, since such events have been found to occur at different frequencies at different genomic sites. Mutations at some sites may be beneficial by increasing genetic diversity while at other sites may adversely affect the survival of an organism. Different DNA sequences and structures exert different masking effects for enzymes (glycosylases) to recognize and repair damages. DNA replicating enzymes, the polymerases may also function inaccurately at certain sites. This study should provide fundamental information on how mutations occur in the genome. Educational activities center around using this research and discovery as tools to stimulate learning and to reinforce concepts. This will be accomplished by including both undergraduate and graduate students in research, and by P.I.'s participation in the revision of an undergraduate biochemistry laboratory curriculum that facilitates discovery based learning, and in two multidisciplinary research training programs. ***
